Portable Thermal Lens Spectrometer with Focusing System

Dec 10, 2004 - Central Research Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan, and Digital Stream Corporation, ...
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Anal. Chem. 2005, 77, 687-692

Technical Notes

Portable Thermal Lens Spectrometer with Focusing System Kazuma Mawatari,†,‡ Yoshiaki Naganuma,§ and Koji Shimoide*,†

Central Research Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan, and Digital Stream Corporation, 4-50-40 Kamitsuruma-Honcho, Sagamihara, Kanagawa 228-0818, Japan

A portable thermal lens spectrometer with a precise focusing system was developed. Astigmatism of the reflected excitation beam from the microchip was used for depth direction focusing. For width direction focusing, the scattering effect of the transmitted probe beam by a microchannel edge was used. The focusing system was evaluated with a 250 µm wide × 50 µm deep microchannel. Focusing resolutions for depth and width directions were 1 and 10 µm, respectively. The repeatability of the thermal lens signal (40 µM xylenecyanol solution) was proved to be ∼1% coefficient of variance when using these focusing methods. The limit of detection for a xylenecyanol solution was 30 nM, and the absorbance was 4.7 × 10-6 AU. The sensitivity was 20-100 times higher than that obtained by spectrophotometry. In consequence, a practical thermal lens spectrometer was realized. The integration of a chemical system on a microchip, which is often called a micro total analysis system (µ-TAS) or a lab-ona-chip, is attracting great interest due to the unique characteristics of the microspace. Application fields range widely including chemical engineering, environmental, and biochemical fields. To realize a totally miniaturized system, detection, microfluidics, and microfabrication technologies are key components. In particular, versatile and highly sensitive detection technologies are needed due to the small volume of the sample in a microchannel. Usually, spectroscopic detection methods are used for on-chip detection. The most sensitive one is the laser-induced fluorescence (LIF) method. For example, single-molecule detection in a microchannel has already been reported.1 However, most molecules exhibit low fluorescence yield and cannot be detected by the LIF method. Spectrophotometry has wide applicability, but its sensitivity is quite low for measurements in a microchannel * To whom all corrspondence should be addressed. E-mail: shimoide.kb@ om.asahi-kasei.co.jp. Fax: +81-545-62-3249. † Asahi Kasei Corp. ‡ Present address: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan. § Digital Stream Corp. (1) Gosch, M.; Blom, H.; Holm, J.; Heino, T.; Rigler, R. Anal. Chem. 2000, 72, 3260-3265. 10.1021/ac049015w CCC: $30.25 Published on Web 12/10/2004

© 2005 American Chemical Society

due to the 1-2 orders shorter optical path length than that of conventional cuvettes. Thermal lens spectrometry (TLS) is a promising method to overcome the low sensitivity of spectrophotometry.2 Since the first report of the thermal lens effect by Gordon et al.,3 TLS has been developed for trace analysis in liquid phase.4-9 Especially, dualbeam TLS (coaxial10,11 or crossed beam12 geometry) offers higher sensitivity than the single-beam TLS. In coaxial dual-beam TLS, the sensitivity is enhanced by the sharp temperature distribution.2 This situation is realized by tightly focusing the excitation and probe beams with a high numerical aperture objective lens. In addition, a focus position mismatch between the excitation and probe beams is important for sensitivity enhancement.13 Kitamori’s group reported the first coaxial dual-beam thermal lens microscope (TLM), which utilized an objective lens with chromatic aberration.14,15 Additionally, sensitive thermal lens detection on the microchip has been realized16,17 and successfully applied to various analytical fields.18-22 (2) Bialkowski, S. E. Photothermal Spectroscopy Methods for Chemical Analysis; John Wiley & Sons: New York, 1996. (3) Gordon, J. P.; Leite, R. C. C.; Moore, R. S.; Porto, S. P. S.; Whinnery, J. R. J. Appl. Phys. 1965, 36, 3. (4) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1979, 51, 728-731. (5) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1980, 52, 2338-2342. (6) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1980, 52, 695A-706A. (7) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1981, 53, 689-692. (8) Haushalter, J. P.; Morris, M. D. Appl. Spectrosc. 1980, 34, 445-447. (9) Skogerboe, K. J.; Yeung, E. S. Anal. Chem. 1986, 58, 1014-1048. (10) Imasaka, T.; Miyaishi, K.; Ishibashi, N. Anal. Chim. Acta 1980, 115, 407410. (11) Buffett C. E.; Morris M. D. Anal. Chem. 1982, 54, 1824-1825. (12) Nolan, T. J.; Dovichi, N. J. Anal. Chem. 1986, 59, 2803-2805. (13) Berthoud, T.; Delorme, N.; Mauchien, P. Anal. Chem. 1985, 57, 12161219. (14) Uchiyama, K.; Hibara, A.; Kimura, H.; Sawada, T.; Kitamori, T. Jpn. J. Appl. Phys. 2000, 39, 5316-5322. (15) Kitamori, T.; Tokeshi, M.; Hibara, A.; Sato, K. Anal. Chem. 2004, 76, 52A60A. (16) Tokeshi, M.; Uchida, M.; Hibara, A.; Sawada, T.; Kitamori, T. Anal. Chem. 2001, 73, 2112-2116. (17) Mawatari, K.; Kitamori, T.; Sawada, T. Anal. Chem. 1998, 70, 50375041. (18) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Nakao, M.; Kitamori T. Anal. Chem. 2000, 72, 1144-1147. (19) Tokeshi, M.; Minagawa, T.; Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Kitamori, T. Anal. Chem. 2002, 74, 1565-1571. (20) Hisamoto, H.; Horiuchi, T.; Uchiyama, K.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 5551-5556.

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Figure 1. Principle of focusing the excitation beam on the bottom interface of the microchannel. Cases are for focusing (a) just on the interface, (b) below the interface, and (c) beyond the interface. The dotted line is the ray refracted by the cylindrical lens, and the solid line is the ray passing through the cylindrical lens without refraction. The letters A, B, C and D denote output signals from each element of the quadrant photodiode.

The conventional TLM was a microscope-based system and was not small enough to serve as a practical system for µ-TAS. Therefore, miniaturization of the TLM system was necessary to realize a totally miniaturized and practical system. Several reports have described this miniaturization. Hiki et al. reported a desktopsized TLM.23 Hattori et al. presented an optical fiber-based system with a rod lens as a palmtop-sized system.24 However, the microscope function to observe the detection position was lost in developing the palmtop-sized system. In the thermal lens measurement, precise focusing of the excitation beam in the center of the microchannel is needed to improve the accuracy of the signal intensity. As shown in Results and Discussion, the signal intensity changes 1-2% for a 2-µm deviation of focus position in the depth direction and it changes 50% for a 17-µm deviation (depth of the microchannel, 50 µm; numerical aperture, 0.4). In addition to the precise focusing, a practical autofocusing system is needed. Therefore, the focus positions of the beams in the microchannel should be quantitatively determined and adjusted. So far, no methods that meet these two demands have been reported. In this work, we integrated a precise and quantitative focusing system with the thermal lens spectrometer. We used astigmatism (21) Sorouraddin, H. M.; Hibara, A.; Proskurnin, M. A.; Kitamori, T. Anal. Sci. 2000, 16, 1033-1037. (22) Shimoide, K.; Mawatari, K.; Mukaiyama, S.; Fukui, H. In Proceedings of Micro Total Analysis System 2002; Baba, Y.; Shoji, S.; van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2002; pp 918921. (23) Hiki, S.; Tokeshi, M.; Hibara, A.; Kitamori, T. Bunseki Kagaku 2003, 52, 569-574. (24) Hattori, A.; Yamaguchi, H.; Yamaguchi, J.; Matsuoka, Y.; Kanki, S.; Fukuzawa, T.; Miwa, T.; Toyama, M.; Tokeshi, M.; Kitamori, T. In Proceedings of Micro Total Analysis System 2003; Northrup, M. A.; Jensen, K. F.; Harrison, D. J., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2003; pp 359-362.

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of the reflected excitation beam for depth direction focusing and used scattering of the transmitted probe beam for width direction focusing. By realizing the focusing system without a microscope, we constructed a practical miniaturized thermal lens spectrometer and evaluated its performance. EXPERIMENTAL SECTION Principles of Focusing. The principle for focusing in the depth direction is illustrated in Figure 1. It is based on astigmatism of the reflected excitation beam from the interface of the microchannel. The basic principle is well known and has been applied for optical disk drives.25-27 When the excitation beam is focused just on the interface (Figure 1a), the reflected beam is completely collimated by the objective lens. A convex lens and cylindrical lens focus the excitation beam on two different positions. The quadrant photodiode is set at the center of the two focus positions. Then, the shape of the beam at the quadrant photodiode becomes round and the calculated output signal is zero as shown. However, when the excitation beam is focused below the interface (Figure 1b), the reflected beam is converged by the objective lens. Then, the two focus positions move closer to the cylindrical lens. As a consequence, the beam at the quadrant photodiode becomes elliptical. In this case, the calculated output signal becomes negative as shown. The same explanation can be applied when the excitation beam is focused beyond the interface (Figure 1c) excluding the two focus positions and the positive output signal. Therefore, the focus position in the depth direction of the microchannel can be adjusted at the middle of the bottom and upper interfaces. (25) Bouwhuis, G.; Braat, J. J. M. Appl. Opt. 1978, 17, 1993-2000. (26) Cohen, D. K.; Gee, W. H.; Ludeke, M.; Lewkowicz, J. Appl. Opt. 1984, 23, 565-570. (27) Mansuripur, M. Appl. Opt. 1987, 26, 3981-3986.

Figure 2. Principle of focusing the probe beam on the edge of the microchannel.

Figure 2 illustrates the principle for focusing in the width direction of the microchannel. It is based on scattering of the probe beam at the edge of the microchannel. When the focus position of the probe beam is set at the edge of the microchannel, the transmitted probe beam is scattered and the transmittance decreases. The edge of the microchannel is determined by measuring the intensity of the transmitted probe beam by a photodiode. Therefore, the focus position in the width direction can be adjusted at the middle of the two edges. The focusing resolutions of these two methods are mainly determined by laser power, numerical aperture of the objective lens, and difference of refractive index between the glass substrate and solution (higher values are desired for all parameters). Optics. An optical diagram of the thermal lens spectrometer is given in Figure 3. (Numbers used in the component descriptions

are as given in the figure.) One laser diode (LD) 1 (Sharp Corp., LT051PS, wavelength 635 nm, output 30 mW) generated an excitation beam. The excitation beam was collimated by the collimator lens 1. The cross-section shape was made round with the prism pair 1 (3 times magnification for the short axis of the ellipse). Another LD 2 (Mitsubishi Electric Corp., ML60114, wavelength 780 nm, output 50 mW) generated a probe beam. The probe beam was collimated by the collimator lens 2. The crosssection shape was made round with the prism pair 2 (2.6 times magnification for the short axis of the ellipse). A beam expander was used to adjust the focus position to enhance the sensitivity of the thermal lens signal.13 Actually, the probe beam was adjusted to focus it 20 µm (the value in air) closer to the objective lens 1 than the excitation beam. Filter 1 (Melles Griot KK, 03FCG111) was used to remove the excitation beam and consequently lowered the background signal. The excitation and probe beams were made coaxial with the beam splitter 1 (100% reflectance for the s-polarized excitation beam, 100% transmittance for the p-polarized probe beam). Then, both beams were introduced into the beam splitter 2 (80% transmittance and 20% reflectance for both excitation and probe beams). The transmitted beams were reflected with the mirror prism 1 and made circular polarized by the λ/4 plate 1. The beams were focused into the microchannel on a microchip by objective lens 1 (Edmund Optics Japan Co., Ltd., 350022B-E, numerical aperture 0.4). The power of the excitation and probe beams were 4.2 and 3.1 mW, respectively, after passing through objective lens 1. In the microchannel, a thermal lens effect was induced and the probe beam was diverged by the effect. The two transmitted beams were collimated by the objective lens 2 (same as objective lens 1) and again made linear polarized with the λ/4 plate 2; only the directions of the polarization were changed by 90°. These beams were reflected with the mirror prism

Figure 3. Illustration of the optical arrangement of the thermal lens spectrometer.

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Figure 4. Illustration of the electronic arrangement of the thermal lens spectrometer.

2. Only the probe beam was reflected by the beam splitter 3 (same as beam splitter 1). The center portion of the probe beam was transmitted using an iris (Edmund Optics Japan Co., Ltd, 53906D) and focused on the quadrant photodiode (PD) 2 (Hamamatsu Photonics KK, S6344) by a lens. The output signal was used for the thermal lens signal and focusing signal in the width direction of the microchannel. The reflected excitation and probe beams from the microchip were again introduced into the beam splitter 2, and 20% of the power was reflected. The probe beam was cut by the filter 2 (Melles Griot KK., 03FIL250), and only the excitation beam was focused on the PD 1 (same as PD 2) by a combination lens (focal length 20 and -12 mm) and a cylindrical lens (focal length 286 mm). The output signal from the PD 1 was used as the focusing signal in the depth direction of the microchannel. The beam splitter 1 and λ/4 plate 1 also worked as an optical isolator to prevent reflected beams from the optical components and the microchip returning to the same LD. All the optical components were fixed in two cases by using a UV-cured adhesive after adjusting their positions. The cases (focusing pickup and detection pickup) were each 15 cm × 10 cm × 3 cm. Electronics. The electronic diagram of the thermal lens spectrometer is given in Figure 4. Both LD 1 and LD 2 were controlled by LD drivers (Asahi Data Systems Ltd., ALP-6323CA, autopower control mode). A function generator (Hewlett-Packard, 8116A) was used to modulate the excitation beam intensity at 1.1 kHz, which was determined from the signal-to-noise ratio (S/N) of the thermal lens signal. One of the output signals from PD 2 was amplified by a preamplifier (NF Corp., LI-57A, 100 times amplification) and fed into a lock-in amplifier (NF Corp., 5610B). The output signal from the lock-in amplifier was recorded as the thermal lens signal. The other output signal from PD 2 was used as a focusing signal in the width direction of the microchannel. The output signal from PD 1 was used as the focusing signal in the depth direction. All the signals were sent to a personal computer (PC) and recorded. The microchip was set on a remotecontrol stage and could be moved in two directions with high resolution (0.1 µm in depth direction and 1 µm in width direction of the microchannel) by the PC. 690

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Microchip. The fabrication method of the microchip was reported previously.28 Three glass plates made of fused silica, a cover plate (500 µm thick), a middle plate (50 µm thick), and a bottom plate (500 µm thick), were bonded to make the microchip (50 mm × 40 mm). The straight and rectangular sectioned microchannel (250 µm wide × 50 µm deep × 30 mm long) was formed in the middle plate. Holes were formed in the cover plate by which the sample solutions could be introduced into the microchannel. RESULTS AND DISCUSSION Thermal lens signal is sensitive to the focus position of the beam, especially in the depth direction. For highly sensitive detection in the microchannel, a tightly focused laser beam is needed to enhance the sensitivity; that is, a more accurate focusing system is needed. The thermal lens signal was measured by changing the position of the microchip in the depth direction in order to investigate the dependence. The sample was a xylenecyanol solution of 40 µM. The numerical aperture of the objective lens was 0.4. The result is shown in Figure 5. At first, the signal intensity was confirmed to linearly increase with the power of excitation beam in the range of 0.4-5 mW. The signal intensity decreased 1-2% from the maximum intensity for a 2-µm change of the focus position from the center of the microchannel. For the 17-µm focus position change, the signal decreased ∼50% from the maximum. The full width at half-maximum (fwhm) was estimated to be ∼34 µm. For applications such as environmental analysis and medical diagnosis, ∼1% accuracy for the coefficient of variance (CV) is needed. Therefore, the accuracy of 1 µm is needed for the focus position in the depth direction. Next, the focus position was measured utilizing the system shown in Figure 6. In Figure 6a, the focus position of the excitation beam was scanned in the depth direction of the microchannel. Near the upper and bottom interfaces of the microchannel, the calculated output signals from the quadrant PD 1 were sigmoidshaped curves as expected. Zero-cross points of the sigmoid curves, the positions where the calculated values were zero, corresponded to just focus positions on the interfaces. The (28) Sato, K.; Tokeshi, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 647650.

Figure 5. Dependence of the thermal lens signal on the z-position of the microchip. Sample was 40 µM xylenecyanol solution. The microchannel was designed with a 250 µm width × 50 µm depth.

resolution of the positioning was estimated to be less than 1 µm from the S/N of the curves. Therefore, the value was enough for accurate measurement. The depth of the microchannel was estimated to be 38 µm (50 µm design value) by measuring the distance between the zero-cross points of the curves. A correction is needed for the value due to the difference in the focusing angle of the excitation beam in air and in the solution. The correction coefficient is calculated as

R)

d ) Rd′

(1)

tan(90° - θ′) tan(90° - θ)

(2)

sin θ ) n sin θ′

(3)

where d is the actual depth of the microchannel, d′ is the measured depth of the microchannel by our method, R is a correction coefficient, θ is the angle of incidence in air, and θ′ is the angle of refraction in the solution. From eqs 1-3, the correction coefficient R was calculated to be 1.38 using the refractive index of water of 1.33 and θ of 23.6° derived from the numerical aperture 0.4 of the objective lens 1. Therefore, the actual depth of the microchannel was calculated to be 52 µm. The 4% difference from the design value of 50 µm was within the fabrication error of 10%. In addition, the error of the depth measurement by this method was confirmed to be less than 1% using a standard glass cell (the channel of 2 mm width × 50 µm depth was fabricated with more than 1% precision). From the measurement in the width direction (Figure 6b), the width of the microchannel was estimated to be 243 µm. The 3% difference from the design value of 250 µm was also within the fabrication error of 5%. The resolution was ∼10 µm from the fwhm of the peak and enough for the measurement. To evaluate the focusing methods, the CV value of the thermal lens signal (40 µM xylenecyanol solution) was investigated using the focusing system. The procedure for focusing the excitation beam in the center of the microchannel was as follows. At first, the bottom interface (air/glass) was examined and the focus position of the excitation beam was adjusted onto the interface. Then, the microchip was moved in the depth direction by 365 µm to focus the probe beam in the middle of the microchannel in the depth direction. The value was the sum of the bottom plate

Figure 6. Focusing signals obtained by scanning the microchip (a) in the depth direction and (b) in the width direction.

thickness (500 µm), half-depth of the microchannel (26 µm), and the interval of the focus positions between the probe and excitation beams (20 µm), which were corrected by eqs 1-3 (1.46 was used for the refractive index of the glass). Next, the edges of the microchannel were examined and the focus position of the probe beam was adjusted to the middle of the two edges. Finally, the focus position of the excitation beam was again adjusted to the middle of the microchannel in the depth direction for accurate focusing. At this position, the thermal lens signal was measured. From 10 measurements, ∼1% was obtained as the CV value. As a consequence, the focusing system was shown to have enough resolution and accuracy. Finally, the limit of detection (LOD) was determined. The concentrations of the xylenecyanol solution were changed in the range of 0-40 µM. For each measurement, the focus position was adjusted as described above and the intensity of the thermal lens signal was measured. As a result, the signal intensity linearly depended on the concentration. The blank (pure water) signal intensity was 0.05 mV, and the slope of the calibration curve was 0.6 mV/µM with a correlation coefficient of 0.9995. The signal intensity was 0.09 ( 0.005 mV at the lowest concentration of 60 nM. From the calibration curve, the determination limit (twice the standard deviation 2σ of the calibration curve) was 40 nM. LOD (S/N ) 2) was calculated as 30 nM from the noise level of the signal. The absorbance of 30 nM solution was calculated as Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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4.7 × 10-6 AU (molar absorption coefficient 3.0 × 104 M-1‚cm-1, optical path length 52 µm). The sensitivity was estimated as 20100 times higher than that of spectrophotometry. In measurements on a microchip, detection is sometimes done under flow conditions. Thermal lens signal intensity depends on the flow rate as reported previously.7 However, the sensitivity was constant within the deviation of 1% below an average flow velocity of 1 mm/s. Therefore, the system can be used without the loss of LOD below the value. In conclusion, a thermal lens spectrometer was developed with precise focusing system. The focusing methods can also be applied to autofocusing. In comparison with other small-sized thermal lens spectrometers, the focusing method in the depth direction can be applied only for our system. A reliable palmtop-sized thermal

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lens spectrometer could be constructed without a microscope. As one application, we are now constructing a portable device for point of care clinical diagnosis using this portable TLM and a microchip.22 Reagents for blood analysis by colorimetric detection are widely available for a wavelength of ∼600 nm. Our system is expected to be a powerful and practical analytical tool for µ-TAS. ACKNOWLEDGMENT We gratefully acknowledge Prof. Takehiko Kitamori of The University of Tokyo for useful discussions and suggestions. Received for review July 5, 2004. Accepted October 14, 2004. AC049015W